1. Field of the Invention
The present invention relates to new tetracarboxylate fluorescent calcium indicators which are targeted to specific intracellular environments, locations or compartments. These new indicators include (1) PE3, a zwitterionic indicator which resists leakage and compartmentalization and therefore remains cytosolic and (2) FFP18, an amphipathic indicator which binds to cellular membranes and therefore reports calcium in the vicinity of the membrane. Each of these indicators are derived from a new BAPTA-based chelator moiety which serves as a convenient and flexible starting point for generating a wide variety of new calcium indicators while retaining the ion selectivity and pH insensitivity of BAPTA.
2. Background of the Invention
Many external environmental signals produce changes in cellular activities or behavior by causing a change in the intracellular free calcium concentration. Thus a proper understanding of cell physiology is often criticially dependent on the ability to monitor changes in cellular calcium concentrations. The introduction of fura-2 and indo-1 provided revolutionary new approaches for studying calcium in individual cells. These indicators were much brighter than their predecessor, quin2, and made it possible to measure calcium based on the ratio of fluorescence intensity at two excitation or emission wavelengths. The feasibility of ratio-based measurements quickly stimulated the development of fluorescence ratio fluorometric instruments and imaging systems and has led to a multitude of new findings regarding the spatial and temporal apsects of calcium regulation in cells.
The rapid adoption of fura-2 and its relatives, and the experience gained in using these indicators also quickly revealed several problems related to dye loading, such as leakage or compartmentalization, spectral alterations between intracellular dye and that in in free solution and unwanted binding to cellular constituents. Furthermore, it has become apparent that fura-2 does not always faithfully report rapid changes in calcium seen in some excitable cells and may altogether miss some rapid and/or highly localized calcium transients.
The problem of fura-2 leakage and compartmentalization was first noted in the consistent failure of fura-2/AM to load in PTK1 cells. Efforts to improve loading using Pluronic f127 gave cells that were initially bright but with time, the indicator rapidly leaked out of the cell leaving only perinuclear vesicles that were still brightly fluorescent. Loss of cytosolic indicator was retarded when cells were incubated at lower temperatures, suggesting that some cellular transport system was involved. Additional information was obtained from experiments in which a variety of different tetracarboxylate indicators were iontophoresed into the cytosol of Tradescantia cells (Hepler and Poenie, unpublished). The initial cytosolic fura-2 distribution was obvious because the indicator spread rapidly to adjacent cells. However, within a short time, virtually all of the cytosolic indicator was transferred to the vacuole. This result showed that cells had mechanisms for actively transporting the anionic form of the indicator. The one indicator which differed in this respect was rhod-2, which, unlike the other indicators, contained a positive charge. This suggested that a zwitterionic indicator might be less susceptible to leakage and compartmentalization. Reports that anion transport inhibitors suppressed the leakage and compartmentalization of fura-2 added further support for this hypothesis.
Although keeping cells at lower temperatures or using anion transport inhibitors such as probenecid and sulfinpyrazone can help reduce leakage and compartmentalization, they add additional variables to the experiment. Conjugation of indicators to dextrans provides another method for preventing dye leakage and compartmentalization but these indicators must loaded into cells by microinjection. The use of rhod-2 would seem to be a good solution to the problem but unfortunately, rhod-2 exhibits neither the spectral shift seen with fura-2 nor the large increase in fluorescence exhibited by fluo-3. As a result, it has not been nearly as popular as the other indicators.
Another problem, which has been the subject of considerable dispute, is how faithfully fura-2 tracks cellular calcium transients. Work by Hollingsworth and Baylor suggested that fura-2 was not fast enough to follow rapid changes in muscle calcium and was unable to track calcium levels at their peak. More enigmatic were cases such as the report of Kao, et. al., where a variety of indirect measures indicated the existence of a calcium transient during mitosis but these were seldom seen when monitored with fura-2. While the absence of a calcium transient might be the right result, it also seemed possible that calcium transients were sufficiently fast and/or localized to preclude detection with fura-2. This latter possibility has gained credence with a recent report showing that large but highly localized increases in calcium do occur in cells and these transients are not readily detected using fura-2. To circumvent this problem it was necessary to develop indicators which responded more quickly to changes in calcium and placed them nearer to the source of the calcium increase.